Fluorescent dyes are valuable reagents for the analysis and separation of molecules and for the labeling and detection of biomolecules for in-vitro and in-vivo for research in development of new drugs, pharmacology, pathology and disease detection. Flow cytometry can be used to detect fewer than 10,000 fluorescein dye labeled cells (Muirhead, Horan and Poste, BIOTECHNOLOGY, 3, 337-356 (1985)).
For in-vivo studies, specifically sequenced peptides can be labeled with fluorescent dyes and imaged as circulated through blood and tissue and by concentrated in tumors and other disease sites. Stem cells can be labeled with dyes to follow their development. Nucleotides can be covalently attached to dyes to allow DNA, RNA, and genetic studies. For in-vitro studies, fluorescent dyes can be covalently bound to antibodies and used in proteins assays as Western Blot. Brightly fluorescent dyes have demonstrated utility as labeling reagents for a variety of biological applications, eg U.S. Pat. Nos. 4,981,977 to Southwick, et al. (1991); 5,286,486 to Waggoner et al. (1993); 5,569,587 to Waggoner (1996); 5,627,027 to Waggoner (1997); 5,808,044 to Brush, et al. (1998); 5,877,310 to Reddington, et al. (1999); 6,008,373 to Waggoner, et al. (1999); 6,043,025 to Minden, et al. (2000); 6,127,134 to Minden, et al. (2000); 6,130,094 to Waggoner, et al. (2000); 6,133,445 to Waggoner, et al. (2000); also WO 97/40104, WO99/51702, WO 01/212624, and EP 1 065 250 A1; and TETRAHEDRON LETTERS 41, 9185-88 (2000); all of the above incorporated by reference.
Nevertheless many carbocyanine dyes are known to share certain constraints for the above purposes. One constraint is the absorbtion and emission characteristics of the fluorescent dye, since many ligands, receptors, and materials in the sample under test, e.g. blood, tissue, bone, will fluoresce and interfere with an accurate determination of the fluorescence of the dye. This phenomenon is called autofluorescencse or background fluorescence.
Another consideration is the ability to conjugate the fluorescent dye to ligands and receptors and other biological and non-biological materials and the effect of such conjugation on the fluorescent dye. In many situations, conjugation to another molecule may result in a substantial change in the fluorescent characteristics of the fluorescent dye and in some cases, substantially destroy or reduce the quantum efficiency of the fluorescent dye. It is also possible that conjugation with the fluorescent dye will inactivate the function of the molecule that is labeled.
Another concern is whether there is non-specific binding of the fluorescent dyes to other compounds or container wall, either by themselves or in conjugation with the compound to which the dye is attached.
In addition certain desired sulfoalkyl derivatives of the reactive carbocyanine dyes are difficult to prepare, indicated in Cy3 and Cy5 variants by Waggoner and colleagues in BIOCONJUGATE CHEM., 4, 105-109 (1993). Cyanine dyes also have very strong tendency to self-aggregate which can significantly reduce the fluorescence quantum yields as described by Mishra, et al., CHEM REV., 100, 1973 (2000). The self-aggregation often limits the amount of dye that can be attached to the desired biomolecule which in turn limits the brightness that can be achieved per conjugate. Another concern of carbocyanine dyes is their sensitivity to destruction by light and oxygen such that handling, storage and testing of the dyes and their conjugates often requires protecting them from light.
Since all of these factors must be considered in design a useful dye for bio-applications, there is a desire to make the dye as useful as possible in the equipment that measures the dyes. One consideration is the emissive capability of the dye often measured by its quantum efficiency. Another consideration is the light absorbing capability of the dye often measured by its extinction coefficient, which should be as large as possible.
Typical fluorescent dyes in use as labeling reagents for biological molecules such as xanthenes, dipyrrometheneboron difluorides, rhodamines and carbocyanines commonly have Stoke shifts of less than about 30 nm. Here the Stoke shift is defined as the difference between the fluorophore's peak excitation and peak emission wavelengths. Because the optimum wavelength of the exciting light is close to close to the peak emission light, dyes with small Stoke shifts require precise excitation and emission filters to eliminate or reduce interference. Furthermore, the customary use of excitation and emission bandpass filters means that only a fraction of the available excitation and emission light may be practically utilized for dyes possessing a small Stoke shift resulting in a diminution of the fluorescence signal. Fluorescent materials that incorporate bright fluorescent dyes with increased Stoke shift permit maximum utilization of the available excitation and emission light, resulting in a greater fluorescence signal.
Another advantage of fluorescent materials with large Stoke shifts is that they can be more easily detected in the presence of other fluorescent materials. For example, immunoassays are typically carried out in body fluids which contain may endogenous fluorescent molecules, such as bilins, flavins and drugs. Since the vast majority of interfering fluorescent materials have relatively short Stoke shifts, the use of a fluorescent label that emits at a wavelength far greater than its excitation wavelength makes the label easier to distinguish from background fluorescence, since its fluorescent signal is emitted at a wavelength at which most background fluorescence is minimal.
A third advantage of fluorescent materials with large Stoke shifts is their usefulness in detecting multiple analytes in a single sample using a single excitation wavelength. Using two or more different fluorescent labels, each of which can be excited at a particular wavelength, the emission peaks of the different labels are detected at different wavelengths, where each emission spectrum is characteristic of a single analyte. In order to successfully accomplish this, the emission peaks of the fluorescent labels must be well-separated from each other so the correction factors between the various dyes are minimized. Fluorescent materials with a large Stoke shift can be used in combination with fluorescent materials with a smaller Stoke shift where both materials excite at the same wavelength, but emit at different wavelengths, giving multiple signals that can be resolved using optical filters or monochromators.
Unfortunately, fluorescent compounds useful as labeling agents that have Stoke shifts of 50-100 nm, or more, as well as high fluorescence efficiency and emission wave lengths of greater than 500 nm required for detectability are relatively rare (Haughland, Fluorescein Substitutes for Microscopy and Imaging, Optical Microscopy for Biology pp. 143-57, 1990). The magnitude of the Stoke shift in fluorescent dyes has been found to be generally inversely proportional to the high absorbance needed to ensure a strong fluorescence signal. Fluorescent dyes in use as labelling reagents for biological molecules commonly have Stoke shifts of less than about 30 nm.
The lack of suitable fluorescent dyes with large Stoke shifts has led to the development and use of protein-based fluorophores known as phycobiliproteins as labels (e.g. U.S. Pat. Nos. 4,520,110 and 4,542,104). Like other fluorophores, they have been covalently attached to beads and macromolecules (see, for example, Oi et al., J. Cell Bio., 93, 981, 1982). These large bilin-containing molecules have the desirable characteristics of very high extinction coefficients and they use internal energy transfer between multiple, unlike, covalently-linked fluorophores to accomplish a relatively large Stoke shift. They have the disadvantage of poor chemical stability, poor photostability, limited long wavelength emission capability, bulky molecular size (MW>100,000 Daltons) and relatively high cost. Furthermore, only a few proteins of this type are known and one cannot select or appreciably adjust their spectral properties. In an effort to improve the fluorescent emission efficiency of phycobiliproteins without significantly increasing their molecular size, they have been covalently coupled to the fluorescent dye Azure A (U.S. Pat. No. 4,666,862).
In studies of energy transfer between pairs of covalently linked dyes, it has been shown that the efficiency of energy transfer between two fluorescent dyes is inversely proportional to the sixth power of the distance between the two interacting molecules, consistent with Fbrster's theory (Stryer and Haughland, Energy Transfer: A Spectroscopic Ruler, Proc. Nat'l Acad. Sci., USA, 58, 719, 1967). The reference suggests that the percentage of measurable energy transfer can be used to measure the distance separating the covalently linked fluorophores in the 10 to 60 Å range. A subsequent paper (Haughland et al., Dependence of the Kinetics of Singlet-Singlet Energy Transfer on Spectral Overlap, PNAS, 63, 23, 1969) reported that intramolecular singlet energy transfer also depends on the magnitude of spectral overlap integral between the emission spectrum of the donor dye and the excitation (absorbance) spectrum of the acceptor dye.
It is known that covalent coupling of a pair of fluorophores in accordance with Förster's theory can result in a fluorescent dye with a larger Stoke shift than either of the individual dyes (e.g. Gorelenko et al., Photonics of Bichromophores Based in Laser Dyes in Solutions and Polymers, Exp. Technik der Physik 37, 343, 1989). This approach, although reportedly effective in increasing the Stoke shift, requires complex synthetic procedures to chemically couple the two dyes together and are limited by the number and location of available reactive sites. The process of carrying out the necessary synthetic procedures to attach multiple dyes sufficiently close together and in the proper spatial and orientational configuration to undergo substantial energy transfer would be exceedingly difficult, if not impossible. Furthermore, covalently linked molecules typically have sufficient freedom of movement that significant collisional deactivation of the excited state occurs, leading to loss of energy by vibrational relaxation rather than by fluorescence. Energy transfer with resultant wavelength shifting has also been described for mixtures of dyes in lasing solutions (e.g. Saito et al., Appl. Phys. Lett., 56, 811, 1990).
The concept of utilizing Förster resonance energy transfer between two or more particle-incorporated fluorescent dyes to achieve an enhanced Stoke shift has been described in U.S. Pat. No. 5,326,692 by Brinkley et al. and U.S. Pat. No. 6,238,931 B1 by Buechler et al.
WO2006/019775 by Pandey, et al., discloses a compound having preferential localization in tumor tissue relative to normal tissue, a preferential electromagnetic absorbtion at a wavelength between about 660 and 900, and a fluorescence at a wavelength shifted from the preferential absorbtion by at least 30 nm. This is accomplished by the combination of two chromophores, one of which is a weakly absorbing tetrapyrrole compound the such that the maximum excitation energy is not shifted from the maximum emission peak by greater than 50 nm
WO2002/32464 by Achilefu, describes a benzindole of the formula:
This structure varies from the current invention in that A6, B6, D6, E6 may together form a 6 or 7 membered carbocyclic ring or a 6 or 7 membered heterocylic ring optionally containing one or more oxygen nitrogen or sulfur atoms. V6 is a single bond or selected from a group of —O—, —S—, —Se—, —NRa, where Ra is selected from a list of substituents. There is no disclosure of large Stoke shift dyes in this application.
According to WO93/23492 “Unfortunately, fluorescent compounds useful as labeling reagents that have Stoke Shifts of 50-100 nm or more, as well as high fluorescence efficiency and emission wavelengths of greater than 500 nm required for detectability are relatively rare. Fluorescent dyes in-vivo as labeling reagents for biological molecules, such as xanthenes, bipyrrometheneboron difluorides, rhodamines and carbocyanines commonly have Stoke Shifts of less than about 30 nm. To solve this problem, multiple fluorescent dyes were immobilized in a polymeric matrix. However the attachment of polymeric matrix of fluorescent dyes to biological molecules requires increased synthetic complexity and the polymeric materials can have adverse effects on biodistribution, clearance, cellular uptake, and cytotoxicity.
U.S. Pat. No. 5,573,909 relates to methods for labeling or detecting one or more target materials using surface coated fluorescent microparticles with unique characteristics. The unique microparticles have at least two components: an external substance or coating that is selective for each target material and an internal mixture of multiple fluorescent dyes. The mixture of dyes is a series of two or more fluorescent dyes having overlapping excitation and emission spectra allowing efficient energy transfer from the excitation wavelength of the first dye in the series, and resulting in fluorescent microparticles with a desired effective Stoke shift, transfer through the dyes in the series and re-emitted as an optical signal at the emission wavelength of last dye in the series, resulting in a desired effective Stoke shift for the microparticle that is controlled through selection of appropriate dyes. The unique microparticles are combined with a sample thought to contain the target material(s), so that the microparticles label the target materials. The sample is then optionally illuminated, resulting in fluorescence of the microparticles that is used to detect one or more target materials.
The invention relates to methods of labeling target materials using fluorescent microparticles having a surface coating that is selective for the target materials.
U.S. Pat. No. 5,326,692 relates to microparticles incorporating a series of two or more fluorescent dyes having overlapping excitation and emission spectra allowing efficient energy transfer from the excitation wavelength of the first dye in the series, transfer through the dyes in the series and re-emitted as an optical signal at the emission wavelength of last dye in the series, resulting in a desired effective Stoke shift which is controlled through selection of appropriate dyes. The novel fluorescent microparticles are useful in applications such as the detection and analysis of biomolecules, such as DNA and RNA, that require a very high sensitivity and in flow cytometric and microscopy analytical techniques.